Taq Thermo Scientific Phusion DNA...

Thermo Scientific Phusion DNA PolymerasesPowerful, accurate, and fast polymerases for better PCR

Find out more at thermofi sher.com/phusionFor Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientifi c Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientifi c and its subsidiaries unless otherwise specifi ed. Affi body is a trademark of Affi body AB. PfuUltra is a trademark of Agilent Technologies, Inc. FastStart and Expand are trademarks of Roche Diagnostics, Inc. CO126872 1115

Engineered polymerase for uracil-tolerant PCRProofreading DNA polymerases are unable to amplify uracil-containing templates due to a so-called uracil-binding pocket, which detects uracil residues in the template strand and stalls further DNA synthesis. Thermo Scientifi c™ Phusion™ U DNA Polymerase carries a mutation in the uracil-binding pocket to overcome this limitation.

Thermo Scientifi c™ Phusion™ U Hot Start DNA Polymerase retains all features of Phusion family enzymes—great accuracy, speed, ability to amplify long amplicons up to 20 kb, and a high specifi city with Affi body ligand–based hot start.

Applications• Amplifi cation of bisulfi te-converted DNA• Amplifi cation of damaged or aged DNA• Carryover contamination control• Uracil excision–based (USER) cloning methods

Figure 8. Highest yields and specifi city with uracil-containingtemplates. Five proofreading DNA polymerases and hot start Taq polymerase were used to amplify a 798 bp fragment of bisulfi te-treated human genomic DNA. Phusion U Hot Start DNA Polymerase provided high yields of specifi c products, whereas all other enzymes delivered zero or lower yields, with some also amplifying products nonspecifi cally.

Figure 9. Highly multiplexed PCR in the shortest time. 19 fragments (73–2,527 bp) from human genomic DNA were simultaneously amplifi ed using different multiplex PCR master mixes according to manufacturers’ recommendations. Phusion U Green Multiplex PCR Master Mix enabled amplifi cation of all 19 fragments and resulted in the fastest PCR protocol.

Maximum performance in multiplex PCRThermo Scientifi c™ Phusion™ U Multiplex PCR Master Mix supports simultaneous amplifi cation of multiple targets up to 2.5 kb over a wide range of template concentration and GC content. The master mix allows fast, sensitive, and inhibitor-tolerant multiplex PCR on any type of template DNA.

Applications • Genotyping • Pathogen detection• Food testing• Analysis of genetically modifi ed organisms• Amplifi cation of microsatellites

Enzyme characteristics and formats

Phusion High-Fidelity DNA Polymerase

Phusion Hot Start II High-Fidelity DNA

Polymerase

Phusion Flash High-Fidelity DNA

Polymerase

Phusion U Hot Start DNA Polymerase

Phusion U Multiplex PCR Master Mix

Blunt or 3’A end Blunt Blunt Blunt Blunt Blunt

Target length, genomic/phage DNA ≤16/20 kb ≤16/20 kb ≤16/20 kb ≤20 kb ≤2.5 kb

Hot start No Yes Yes Yes Yes

Recommended extension time 15–30 s/kb 15–30 s/kb 15 s/kb 15–30 s/kb 15–30 s/kb

Fidelity vs. Taq 52x 52x 25x 25x NA

dUTP tolerance No No No Yes Yes

Enzyme1 ü ü – ü –Green Buffer2 ü ü – ü üMaster mix3 ü ü ü ü üComplete kit 4 ü – – – –

1. DNA polymerase, buffer(s), DMSO, and MgCl2

2. DNA polymerase supplied with Green Buffer, which includes density reagent and two tracking dyes for direct loading on gel

3. 2X master mix

4. All the necessary PCR reaction components including control template and primers

Ordering information

Product Quantity Cat. No.

Phusion High-Fidelity DNA Polymerases and Master Mixes

Phusion High-Fidelity DNA Polymerase100 U F-530S

500 U F-530L

Phusion Green High-Fidelity DNA Polymerase 100 U F-534S

500 U F-534L

Phusion High-Fidelity PCR Master Mix with HF Buffer

100 x 50 µL rxns F-531S

500 x 50 µL rxns F-531L

Phusion High-Fidelity PCR Master Mix with GC Buffer

100 x 50 µL rxns F-532S

500 x 50 µL rxns F-532L

Phusion High-Fidelity PCR Kit50 x 50 µL rxns F-553S

200 x 50 µl rxns F-553L

Phusion Hot Start II High-Fidelity DNA Polymerase100 U F-549S

500 U F-549L

Phusion Green Hot Start II High-Fidelity DNA Polymerase

100 U F-537S

500 U F-537L

Phusion Hot Start II High-Fidelity PCR Master Mix100 x 50 µL rxns F-565S

500 x 50 µL rxns F-565L

Phusion Green Hot Start II High-Fidelity PCR Master Mix

100 x 50 µL rxns F-566S

500 x 50 µL rxns F-566L

Phusion Flash High-Fidelity PCR Master Mix100 x 20 µL rxns F-548S

500 x 20 µL rxns F-548L

Phusion U DNA Polymerases and Master Mixes

Phusion U Hot Start DNA Polymerase100 U F-555S

500 U F-555L

Phusion U Green Hot Start DNA Polymerase100 U F-556S

500 U F-556L

Phusion U Hot Start PCR Master Mix100 x 50 µL rxns F-533S

500 x 50 µL rxns F-533L

Multiplex PCR Master Mixes

Phusion U Multiplex PCR Master Mix100 x 50 µL rxns F-562S

500 x 50 µL rxns F-562L

Phusion U Green Multiplex PCR Master Mix100 x 50 µL rxns F-564S

500 x 50 µL rxns F-564L

Other Phusion Polymerase–based products

Phusion Site-Directed Mutagenesis Kit 20 rxns F-541

References1. Gibson DG et al. (2008) Complete chemical synthesis, assembly, and cloning of a

Mycoplasma genitalium genome. Science 319:1215–1220.

2. Gibson DG et al. (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52–56.

3. Kinde I et al. (2011) Detection and quantifi cation of rare mutations with massively parallel sequencing. PNAS 108:9530–9535.

4. Frey B, Suppmann B (1995) Demonstration of the Expand PCR System’s greater fi delity and higher yields with a lac I-based PCR fi delity assay. Biochemica 2:8–9.

5. Vandenbroucke I et al. (2011) Minor variant detection in amplicons using 454 massive parallel pyrosequencing: experiences and considerations for successful applications. Biotechniques 51:167–177.

“After realizing that we could get the same number of cycles in roughly a quarter of the time (and at only slightly higher per unit cost), we changed exclusively to Phusion [Polymerase].”

PhD studentDepartment of Biological Sciences

Idaho State University, USA

P: Thermo Scientifi c™ Phusion™ U Green Multiplex PCR Master Mix

A–D: Multiplex PCR master mixes from other suppliersM: Thermo Scientifi c™ GeneRuler™ 100 bp Plus DNA Ladder

M P A B C D M

molecular biology

A: Proofreading fusion-type polymeraseB–D: Uracil-tolerant proofreading DNA polymerases from other vendorsE: Hot start Taq DNA polymerase

P: Phusion U Hot Start DNA Polymerase

Upgrade to the gold standard for high-performance PCRSince their introduction in 2003, Thermo Scientific™ Phusion™ High-Fidelity DNA Polymerases have established the gold standard for high-performance PCR. Phusion products are referenced in thousands of publications and have become the first-choice DNA polymerases for a multitude of applications ranging from reconstruction,1 design,2 and massively-parallel, high-throughput sequencing of whole genomes.3

In Phusion High-Fidelity DNA Polymerase, a DNA-binding domain is fused to a Pyrococcus-like proofreading polymerase. Due to this unique fusion technique, Phusion DNA Polymerases generate PCR products with very high accuracy and speed. In addition, Phusion DNA Polymerases are tolerant of various inhibitors, allowing for robust amplification of PCR products with minimal optimization. For hot start PCR, Thermo Scientific™ Phusion™ Hot Start II High-Fidelity DNA Polymerase is an ideal choice allowing high specificity and improved robustness.

The processivity* of Phusion DNA Polymerases is approximately 10-fold greater than that of Pfu DNA polymerase and twice that of Taq DNA polymerase. This high processivity results in shorter extension times, more robust amplification and the ability to amplify long templates (up to 20 kb) in a fraction of the time. Phusion DNA Polymerases also produce higher yields while using less enzyme than traditional proofreading polymerase reactions.

Features

• High fidelity—52x more accurate than Taq, 6x more accurate than Pfu

• Enhanced robustness—fewer reaction failures and minimal optimization

• High speed—increased processivity allows shorter reaction times (extension 15–30 s/kb)

• Improved yields—high product yields with minimal enzyme amounts (0.5–1 U/50 µL reaction)

• Enhanced specificity—unique hot start technology with zero-time reactivation reduces nonspecific amplification and primer degradation

• Simplified workflows—Phusion Green DNA Polymerases allow direct loading of PCR products onto gels

Applications

• High-fidelity PCR• Fast PCR• Hot-start PCR• Long range PCR (up to 20 kb)• High-throughput PCR

The Thermo Scientific™ Phusion™ Green format is a combination of Phusion DNA Polymerases and 5X Green Reaction Buffer. The buffer includes a density reagent and two tracking dyes (blue and yellow) for direct loading of PCR products on gels. The green buffer does not interfere with the performance of Phusion DNA Polymerases and is compatible with downstream applications including DNA sequencing, ligation, and restriction digestion.

To learn more, go to thermofisher.com/phusion

*Processivity measures the number of nucleotides the enzyme can incorporate into a growing DNA strand at one binding event during the extension step.

High fidelityIn many molecular biology applications including cloning, site-directed mutagenesis, and DNA translation, it is crucial to preserve the accurate DNA sequence during PCR amplification. An incorrectly incorporated nucleotide may result in the addition of the wrong amino acid, which, in turn, can affect folding and functional properties of the protein. Alternatively, deletion of a single nucleotide can destroy the correct reading frame.

Phusion DNA Polymerases have very high fidelity. The error rate of Phusion DNA Polymerase as determined by a modified lacI-based method4 is approximately 50-fold lower than that of Taq DNA polymerase and six-fold lower than that of Pfu DNA polymerase (Figure 1).

The low error rate of Phusion DNA Polymerase was confirmed in studies using 454 sequencing5 and Illumina sequencing methods3 (Table 1).

High-specificity hot start PCRThermo Scientific™ Phusion™ Hot Start II High-Fidelity DNA Polymerase combines the Phusion DNA Polymerase and a reversibly bound, specific Affibody™ ligand. The ligand inhibits the polymerase activity at room temperature and thus prevents the amplification of nonspecific products. The reaction set-up can be done at room temperature enabling its use in high-throughput robotics. The Affibody ligand also inhibits the 3´5´ exonuclease activity of the polymerase, preventing degradation of primers and template DNA during reaction set-up. At polymerization temperatures, the ligand is released, rendering the polymerase fully active. Phusion Hot Start II DNA Polymerase does not require a separate activation step in the PCR protocol as it is immediately reactivated at high temperatures.

Robust amplification of GC-rich templatesOptimized buffer system in synergy with the high enzyme processivity enables Phusion DNA Polymerase to amplify a broad range of DNA templates with different sequence content. Efficient amplification can be achieved even with difficult-to-amplify targets, including those with 85% GC content.

Successful amplification of targets up to 20 kbPhusion DNA Polymerases are an ideal choice for amplification of long templates. The high enzyme processivity allows amplification of a wide variety of template sizes. Amplicons up to 20 kb are produced with high yields, short cycling times, and high fidelity.

Fast PCR with high fidelityPhusion DNA Polymerases incorporate a high number of nucleotides per binding event. This high processivity allows extremely short extension times and consequently reduced protocol times. Shortest protocol times can be achieved with Thermo Scientific™ Phusion™ Flash High-Fidelity PCR Master Mix, a product developed specifically for fast PCR.

Better yields with less enzymeDue to their unique structure, Phusion DNA Polymerases are highly efficient and therefore require fewer units per reaction than conventional polymerases. Speed and efficiency result in high product yields in minimal time. In addition, Phusion polymerases are highly robust, minimizing the need for reaction optimization.

Figure 1. Relative fidelity values of different DNA polymerases. Fidelity = 1/error rate.

Phusion DNA Polymerases

Pfu KOD TaqPfu-basedfusion DNApolymerase

52x Taq

32x Taq

8x Taq 7x Taq

1x Taq

a Error rate, number of errors (miscalled bases, inserted or deleted bases) divided by total number of bases.b Dots or Dot, three successive negative flows during 454 sequencing.Table reprinted from Reference 5 with permission of the author and journal.

Table 1. Error rates for different DNA polymerases. Error rates for DNA polymerases were determined by 454 sequencing for following PCR amplification of four different exons from the human TP53 oncogene. The clonal TP53 plasmid was used as a starting template.

KOD (%)Phusion HF (%)

Pt Taq (%)

ExpandHF (%)

FastStartHF (%)

SequalPrep Long (%)

PfuUltraHF (%)

Overall error ratea 0.21 0.11 0.34 0.25 0.23 0.29 0.23

Insertions 0.10 0.07 0.14 0.11 0.11 0.11 0.12

Deletions 0.06 0.02 0.08 0.07 0.05 0.06 0.05

Substitutions 0.01 0.01 0.07 0.04 0.03 0.07 0.01

Dots or Dotb 0.04 0.01 0.05 0.04 0.04 0.05 0.05

Figure 2. High yields of long PCR products. A 20 kb fragment from λ DNA and 7.5 kb fragment from human genomic DNA was amplified with Phusion DNA Polymerases and proofreading DNA polymerases from other suppliers.

PG: Phusion Green High-Fidelity DNA PolymeraseP: Phusion High-Fidelity DNA Polymerase

A– C: Proofreading DNA polymerases from other suppliersM: Thermo Scientific™ ZipRuler™ Express DNA Ladder 2

20 kb (viral) 7.5 kb (genomic)

M PG P A B C PG P A B C M

Figure 3. Higher efficiency with less enzyme. A 3.8 kb fragment from human beta globin gene was amplified with three different DNA polymerases. Phusion DNA Polymerase was able to amplify the 3.8 kb fragment with a combined annealing and extension step of only 1 minute. A single unit of Phusion DNA Polymerase produced higher yields than 2.5 or 5 units of the Pfu DNA polymerases.

Pfu(5 U)

modified Pfu(2.5 U)

Phusion DNA Polymerase (1 U)

Figure 4. High specificity and yields. Five proofreading hot-start DNA polymerases were used to amplify 1.7–2.3 kb fragments from human genomic DNA. Phusion Hot Start II DNA Polymerase provided high yields of specific products, whereas other enzymes delivered zero or low yields, with some also amplifying nonspecific products.

M P A B C D MP A B C D MP A B C D

1.7 kb 2.2 kb 2.3 kb (GC-rich)

P: Phusion Hot Start II High-Fidelity DNA Polymerase

A–D: Proofreading hot start DNA polymerases from other suppliers

Figure 5. Amplification of DNA fragments with different sequence content. Four DNA fragments of different GC content were amplified with different DNA polymerases. Phusion Green DNA Polymerase produced all four amplicons with high yields. In contrast, a competing high-fidelity DNA polymerase was not able to efficiently produce the GC-rich product.

P: Phusion Green High-Fidelity DNA Polymerase

A: Competing high-fidelity DNA polymerase from another supplierM: Thermo Scientific™ FastRuler™ Middle Range DNA Ladder

M P A P A P A P A M

38% 65% 71% 85%

Fast cycler Conventional cycler

Figure 7. High speed and yields with Phusion Flash High-Fidelity PCR Master Mix. A 1.5 kb human cathepsin K gene was amplified with three different polymerases using varying extension times (10–60 seconds). Only Phusion Flash High-Fidelity PCR Master Mix was able to amplify the 1.5 kb gene with very short extension times of 10 and 20 seconds. It also produced superior yields of specific product compared to other enzymes tested.

60 s45 s

30 s20 s

10 s60 s

45 s30 s

20 s10 s

60 s45 s

30 s20 s

Fast Taq polymerasePhusion Flash

Pfu-based fusion polymerase

Figure 6. Shorter PCR run times with Phusion Flash High-Fidelity PCR Master Mix.

Features

Applications

52x Taq

32x Taq

8x Taq 7x Taq

1x Taq

Pt Taq (%)

ExpandHF (%)

FastStartHF (%)

SequalPrep Long (%)

PfuUltraHF (%)

Insertions 0.10 0.07 0.14 0.11 0.11 0.11 0.12

Deletions 0.06 0.02 0.08 0.07 0.05 0.06 0.05

Substitutions 0.01 0.01 0.07 0.04 0.03 0.07 0.01

Dots or Dotb 0.04 0.01 0.05 0.04 0.04 0.05 0.05

Pfu(5 U)

modified Pfu(2.5 U)

M P A P A P A P A M

38% 65% 71% 85%

60 s45 s

30 s20 s

10 s60 s

45 s30 s

20 s10 s

60 s45 s

30 s20 s

Features

Applications

52x Taq

32x Taq

8x Taq 7x Taq

1x Taq

Pt Taq (%)

ExpandHF (%)

FastStartHF (%)

SequalPrep Long (%)

PfuUltraHF (%)

Insertions 0.10 0.07 0.14 0.11 0.11 0.11 0.12

Deletions 0.06 0.02 0.08 0.07 0.05 0.06 0.05

Substitutions 0.01 0.01 0.07 0.04 0.03 0.07 0.01

Dots or Dotb 0.04 0.01 0.05 0.04 0.04 0.05 0.05

Pfu(5 U)

modified Pfu(2.5 U)

M P A P A P A P A M

38% 65% 71% 85%

60 s45 s

30 s20 s

10 s60 s

45 s30 s

20 s10 s

60 s45 s

30 s20 s

Polymerase

3. 2X master mix

500 U F-530L

500 U F-534L

100 x 50 µL rxns F-531S

500 x 50 µL rxns F-531L

100 x 50 µL rxns F-532S

500 x 50 µL rxns F-532L

200 x 50 µl rxns F-553L

500 U F-549L

100 U F-537S

500 U F-537L

500 x 50 µL rxns F-565L

100 x 50 µL rxns F-566S

500 x 50 µL rxns F-566L

500 x 20 µL rxns F-548L

500 U F-555L

500 U F-556L

500 x 50 µL rxns F-533L

500 x 50 µL rxns F-562L

500 x 50 µL rxns F-564L

M P A B C D M

molecular biology

Polymerase

3. 2X master mix

500 U F-530L

500 U F-534L

100 x 50 µL rxns F-531S

500 x 50 µL rxns F-531L

100 x 50 µL rxns F-532S

500 x 50 µL rxns F-532L

200 x 50 µl rxns F-553L

500 U F-549L

100 U F-537S

500 U F-537L

500 x 50 µL rxns F-565L

100 x 50 µL rxns F-566S

500 x 50 µL rxns F-566L

500 x 20 µL rxns F-548L

500 U F-555L

500 U F-556L

500 x 50 µL rxns F-533L

500 x 50 µL rxns F-562L

500 x 50 µL rxns F-564L

M P A B C D M

molecular biology

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